Mycorrhizal association is a nutritional strategy which many plants and some fungi have developed which ensures them a mutual benefit. The mycorrhizae are organs formed by the root of a plant and the mycelium of a fungus. They act as an absorption system which extends through the soil, thus providing a symbiotic relationship that is beneficial both for the plant and for the fungus. In terms of the plant, the growth thereof is facilitated (since the fungus allows the water and the required minerals to be absorbed better, stimulating the growth of the root), improvement in the absorption of phosphate and other nutrients, it is better protected from the toxic effects caused by high concentrations of determined minerals, it resists the lack of water better, it is better protected against the attack of pathogens, the rooting and growth of seedlings is stimulated, it helps to overcome environmental stress situations. In addition, the fungus receives sugars originating from photosynthesis from the plant, essentially starch.
Approximately 90% of all plant species live in symbiosis with a large quantity of fungi in the soil. This form of living in community or symbiosis (which symbolizes the mutual benefit between the plant species and the fungi) is termed mycorrhiza. Since it is such an extensive phenomenon, the term “mycorrhizae” has become the name with which fungi involved in the formation thereof have been designated, although such designation is not very orthodox. The extension of the term and colloquial habits have led to the coining of terms such as “mycorrhize”, that is to say, placing the mycorrhizal fungi into contact with plants or the term “mycorrhization” to indicate the establishment of the symbiosis.
To this end, it is known from the state of the art to use mycorrhizal fungi to provide the plants with the previously mentioned benefits as well as to use pesticide compositions, and for them to facilitate the activity of microorganisms.
Among the documents which have been found in the closest state of the art, there is the Russian patent RU2113794C1, which describes a method for preparing a microbial pesticide with bipolar cover. The described method comprises mixing a biopolymer with water, heating the mixture from 80 to 121° C. up to gel or paste formation, cooling to the temperature from room point to 60° C. The mixture comprises one of the following microorganism strains: Bacillus subtilis krietiensis ATCC 55078 or 55079, Pseudomonas fluorescens ATCC 27663, Gliocladium vixens ATCC 52045, Trichoderma reesei ATCC28217, Trichoderma harzianum ATCC 52445, Trichoderma hamatum ATCC 52198, Trichoderma viride ATCC 52440, Streptomyces cacaoi asoensis ATCC 19093, Bacillus thuringiensis ATCC 13367, Beauveria bassiana ATCC 26851, 48585 or 48023, Hirsutella thomsonii ATCC 24874, Metarhizium flavoviride ATCC 32969, Verticillium lencanii ATCC 46578, Collectotrichum gloeosporioides f. sp. jussiaeas ATCC 52634. The mixture obtained is formed and dried and a pesticide is prepared. Prior to mixing the cooled gel or paste (biopolymer) with the microorganisms or in the process of stabilizing the mixture, the nutrients are expediently added. The following substances may be used as biopolymers (one component), grains, tubers, root crops, starch, wax and natural resins and the derivatives thereof. The stabilizing agents: glycerol, milk, milk.
In addition, the international patent application WO9107869A1 describes a continuous coating for seeds which comprises a liquid formulation in combination with a microorganism to biologically control plant pathogens and a solid material in carbon particle form which includes soft coal, shales, moss, muck soil, activated charcoal or mixtures which protect the seed and the plant from soil-borne pathogens including Pythium spp. The compositions comprise the bioprotective strain Trichoderma 22 (ATCC n° 20847) which is particularly effective at protecting a variety of crops such as tomato, cucumber, cabbage, sugar beet, beetroot, maize field, sweetcorn, cotton, bean, green bean and soya. The composition comprises an adhesive which is within the group composed of polyvinyl alcohol, polyalkyleneoxide, polyvinyl acetate, starch, cellulose esters such as methyl cellulose and hydroxypropyl cellulose, organic silicates, including organic silicon esters, rubbers, alginate and similar.
In order to have the correct interaction between the fungi and the plants, in particular in order to achieve the positive effect of the same in stimulating and germinating the seeds, effective adhesion of the spores to the surface of the same is required which may vary depending on the species of plants and fungi. To this end, it is of great importance to develop a strategy which allows the joining of spores to seeds to be obtained in an effective and stable manner as well as to be able to quantitatively assess the efficacy of said strategy for carrying out anticipated effect monitoring.
Among the different known methods for carrying out monitoring of an anticipated effect, one of the most important methods is electronic microscopy quantification using fluorescent molecular markers that are easily traceable.
Therefore, various methods of microscopy quantification by means of the use of a wide range of fluorescent molecular markers are known in the state of the art. For example, DsRed and GFP (green fluorescent protein) have been used for marking Rhizophagus intraradices using a biolistic approach.
Another example is the transformation of Agrobacterium tumefaciens which has been recently proposed for “in vivo” monitoring of arbuscular mycorrhizal fungi during symbiotic development (Helber and Requena, 2008, New Phytologist, 177: 537 to 548). However, the authors of this publication reported that the expression of fluorescence in transformed spores was transitory and disappeared very quickly. In addition, the efficacy of the transformation mediated by Agrobacterium of spores of the arbuscular mycorrhizal species was very low.
However, to date, no adhesive composition has been described which catalyzes the joining of fungus spores to the surface of vegetable seeds to achieve a stable and effective joining which guarantees the symbiosis phenomenon.
As a result, a method has also not been described in the state of the art which allows the joining of said fungus spores to the surface of seeds to be systematically quantified and monitored by means of fluorescence microscopy in order to prove the efficacy of said joining. Therefore it is necessary to provide a simple and effective method which allows the adhesion of the spores of the fungi to the seeds to be quantitatively evaluated in order to determine the effectiveness of the adhesion method.
The present invention thus relates to an adhesive composition for joining fungus spores to the surface of vegetable seeds in a stable manner which comprises an aqueous solution of fermented maize liquor and/or soya protein hydrolysates.
The present invention also relates to the method for obtaining said adhesive composition as well as to the use of the composition object of the present invention for the adhesion of fungus spores to the surface of vegetable seeds.
The present invention also relates to an indirect method which allows the efficacy of the adhesive composition to be quantitatively assessed characterized in that silica or polyethylene microspheres are used that are fluorescent when they are exposed to ultraviolet (UV) light with similar characteristics to the spores in terms of the size distribution, the spherical shape, density, specific surface and affinity for surface water, owing to which the effectiveness of an adhesive composition intended for adhering fungus spores to vegetable seeds may be determined.
As a result, the present invention provides an adhesive composition of fungus spores to vegetable seeds for promoting or catalyzing the symbiosis phenomenon and the method for verifying or quantifying said adhesion which allows the quality of the adhesive composition to be assessed.
The method is very advantageous since it allows an indirect measurement of the adhesion phenomenon of fungus spores to be obtained, in particular arbuscular mycorrhizae to vegetable seeds which are preferably cereal seeds.
Another advantage of the present invention, in addition to avoiding the use of organisms, in particular genetically modified organisms, is that the fluorescent microspheres present fluorescence in the ultraviolet (UV) spectrum which is a much more stable fluorescence than if arbuscular mycorrhizal spores were used which express fluorescent markers.
The present invention has an important practical application since it provides an adhesive composition for joining fungus spores to the surface of vegetable seeds in a stable manner which comprises an aqueous solution of fermented maize liquor and/or soya protein hydrolysates and it provides a simple and reliable quantitative method for determining the effectiveness of said adhesive composition in the adhesion of fluorescent microspheres to the vegetable seeds and based on the result obtained the effectiveness of said composition for the adhesion of fungus spores, with similar characteristics to the fluorescent microspheres, can be inferred.